The present invention relates to a method and a pharmaceutical composition for disrupting lactation in a mammary gland by reducing the concentration of extracellular divalent calcium cations in the gland, and more particularly, to the use of proteose-peptones (PPs) to accomplish this reduction. The present invention further relates to a method and a pharmaceutical composition for the treatment and prevention of mastitis by reducing the concentration of extracellular divalent calcium cations in infected glands, and more particularly to the use of proteose-peptones to accomplish this reduction.
Citation or identification of any reference in this section or in any other section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
The process of mammary gland involution in ruminants and in other mammals proceeds through several distinct stages that involve cessation of milk production, apoptosis of epithelial cells and tissue remodeling.
During the first stage, mammary involution is triggered by local stimuli that initiate apoptosis, but the process can be reversed by reinitiating milk removal (Capuco and Akers, 1999; Wilde et al., 1999). This local control can cause involution and apoptosis when milk stasis is induced in individual glands, as in lactating goats following unilateral cessation of milking (Quarrie et al., 1994), or in lactating mice by teat-sealing (Li et al., 1997; Marti et al., 1997; Quarrie et al., 1996).
The second stage of involution is persistent, and milk removal can not cause resumption of milk secretion (Capuco and Akers, 1999; Wilde et al, 1999). This stage is characterized by activation of proteases that destroy the lobular-alveolar structure of the gland by degrading the extracellular matrix and basement membrane, as well as massive loss of alveolar cells.
Cellular tight junctions (TJ) form a gasket around the apical side of each epithelial cell joined to a similar structure on adjacent cells. Intact TJ act as a barrier between the apical and basolateral side of the cell, thus preventing paracellular transport and maintaining an electrochemical gradient of up to 35 mV in the mammary gland (Fleet and Peaker, 1978; Nguyen and Neville, 1998). When milk is not continuously removed from the gland, TJ become leaky. With respect to the process of mammary gland involution, it is believed that both local factors, and systemic factors (e.g., prolactin, progesterone, and glucocorticoids) play a role in the regulation of mammary TJ (Nguyen and Neville, 1998).
Maintenance of extracellular Ca+2 levels is essential for maintaining TJ integrity in the mammary secretory epithelium (Neville and Peaker, 1981; Pitelka et al., 1983). Although only about 7% of the total calcium in human and bovine milk is in free ionized form (Neville et al., 1994), this is apparently sufficient to maintain TJ integrity in the intact gland. Neville and Peaker (1981) and Stelwagen et al. (1995) have shown that introducing EGTA into the mammary gland induced transient loss of TJ integrity and transient reduction of milk yield, by acting as a chelator of ionized calcium. The major native proteolytic activity in bovine milk is from the serine protease plasmin and its zymogen (Andrews, 1983). There is evidence suggesting that the plasmin activator (PA)-plasminogen-plasmin system is involved in control of milk secretion and tissue remodeling during involution (Osowski et al., 1979; Politis, 1996).
Proteose-peptones (PPs), also known as caseinophosphopeptides, a group of boiling-resistant peptides constituting about a third of whey proteins, are principally the products of the activity of plasmin on xcex2-casein and alpha-s1- and alpha-s2-casein (Andrews, 1983). Based on point of cleavage of casein moieties by plasmin, about 50% of the protease peptones in whey are phosphopeptides (Anderson et al., 1995). Caseinophosphopeptides have been shown to possess the unique property of being able to bind macroelements such as Ca, Mg, and Fe, along with trace elements such as Zn, Ba, Cr, Ni, Co and Se. One mole of caseinophosphopeptide may bind 20 to 40 mole of Ca, 17 mole of phosphate and 6 mole of Zn (Anderson et al., 1995; Fitzgerald, 1998).
Current management of dairy cows and goats results in significant overlap of lactation and pregnancy, such that these animals are typically pregnant when milking is terminated during late lactation. When milk stasis occurs, the mammogenic and lactogenic stimulation of pregnancy opposes stimuli for mammary involution (Capuco and Akers, 1999). This causes involution of the mammary gland to occur at a slower rate and alveolar structure to be maintained for a greater period of the involution process in dairy cows and goats (Capuco and Akers, 1999; Wilde et al., 1999) than in sheep (Tatarczuch et al., 1997) and rodents (Li et al., 1997).
By accelerating the natural appearance of a local factor which is responsible for inducement of involution, it is possible to override the anti-involution systemic stimuli, and to accelerate the process and extent of involution. Such a capacity to manipulate the process of involution has a great potential to increase animal productivity by enhancing the capacity to control mastitis (Olivier and Sordillio, 1989), and increasing the potential of the cows to produce milk (Capuco and Akers, 1999).
While it is known that milk contains antibiotic/host protective peptides such as casecidin and isracidin (for review see Lahov and Regelson, Food-Chem-Toxicol. (1996) 34(1): 131-45), with apparent efficacy in preventing or reversing infection in livestock animals, the present invention deals with a chemically distinct group of peptides.
Controlling economic losses associated with mastitis in the dairy industry has long been considered the most important single factor in achieving profitability. The increasing prevalence of antibiotic resistant bacteria in the field has made this control increasingly difficult to achieve. Deployment of BST (Bovine Somatotropin) in the milking herd, while increasing overall milk yield, has also contributed to difficulties in controlling mastitis. In many cases, herdsmen must remove high producing animals from the milking herd prior to their scheduled withdrawal in order to treat infection. In addition, mastitis may persist from one lactation cycle to the subsequent one, necessitating the permanent removal of otherwise valuable animals from the milking herd.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method and a pharmaceutical composition to transiently or persistently accelerate the process of involution in one or more glands of a lactating animal. There is also a widely recognized need for, and it would be highly advantageous to have, a method and a pharmaceutical composition for the treatment or prevention of mastitis.
According to one aspect of the present invention there is provided a method of ceasing milk production in a mammary gland of a lactating animal, the method comprising the step of administering a chelating agent into the mammary gland of the lactating animal.
According to another aspect of the present invention there is provided a method of inducing involution in a mammary gland of a lactating animal, the method comprising the step of administering a chelating agent into the mammary gland of the lactating animal.
According to yet another aspect of the present invention there is provided a method of treating an infection in a mammary gland of a lactating animal, the method comprising the step of administering a chelating agent into the mammary gland of the lactating animal.
According to still another aspect of the present invention there is provided a pharmaceutical composition for ceasing milk production in a mammary gland of a lactating animal, the pharmaceutical composition comprising, as an active ingredient, an effective amount of a chelating agent and a pharmaceutically acceptable carrier.
According to an additional aspect of the present invention there is provided a pharmaceutical composition for inducing involution in a mammary gland of a lactating animal, the pharmaceutical composition comprising, as an active ingredient, an effective amount of a chelating agent and a pharmaceutically acceptable carrier.
According to yet an additional aspect of the present invention there is provided a pharmaceutical composition for treating an infection in a mammary gland of a lactating animal, the pharmaceutical composition comprising, as an active ingredient, an effective amount of a chelating agent and a pharmaceutically acceptable carrier.
According to further features in preferred embodiments of the invention described below, the step of administering the chelating agent into the mammary gland of the lactating animal is effected by injecting the chelating agent into a teat canal of the mammary gland of the lactating animal.
According to still further features in the described preferred embodiments, the chelating agent is selected from the group of chelating agents consisting of casein derived proteose-peptones, synthetic phosphopeptides.
According to still further features in the described preferred embodiments, dosing and repetition of the step of administering the chelating agent into the mammary gland of the lactating animal are selected so as to transiently cease milk production in the mammary gland of the lactating animal.
According to still further features in the described preferred embodiments, dosing and repetition of the step of administering the chelating agent into the mammary gland of the lactating animal are selected so as to cause persistent cessation of milk production in the mammary gland of the lactating animal.
According to still further features in the described preferred embodiments, a single dose of protease-peptone in combination with cessation of milking will induce involution and will result in persistent cessation of milk production in the mammary gland of the lactating animal.
According to still further features in the described preferred embodiments, dosing and repetition of the step of administering the chelating agent into the mammary gland of the lactating animal, or a single dose of the chelating agent combined with ceasing of milking are selected so as to cause persistent cessation of milk production in the mammary gland of the lactating animal.
According to still further features in the described preferred embodiments, the animal is a livestock animal.
According to still further features in the described preferred embodiments, the animal is selected from the group of animals consisting of cow, goat, sheep, lama, human, camel, donkey, pig, dog and cat.
According to still further features in the described preferred embodiments, dosing and repetition of the step of administering the chelating agent into the mammary gland of the lactating animal are selected so as cause involution in the mammary gland of the lactating animal which is reversible by ceasing treatment.
According to still further features in the described preferred embodiments, dosing and repetition of the step of administering the chelating agent into the mammary gland of the lactating animal are selected so as cause involution in the mammary gland of the lactating animal which is persistent after ceasing treatment.
According to still further features in the described preferred embodiments, dosing and repetition of the step of administering said chelating agent into the mammary gland of the lactating animal are selected so as to cause involution similar to that which occurs at the end of a lactation cycle in the mammary gland of the lactating animal.
According to yet an additional aspect of the present invention there is provided a method for reducing the amount of free ionic calcium in milk within a lactating mammary gland, the method comprising the steps of (a) administering a proteolytic enzyme into a teat canal, so as to produce at least one proteose peptone by digestion of a substrate within the milk, so as to chelate the free ionic calcium.
According to another additional aspect of the present invention there is provided a pharmaceutical composition for reducing the amount of free ionic calcium in milk within a lactating mammary gland comprising an effective amount of a proteolytic enzyme capable of digesting at least one milk protein so that at least one proteose peptone capable of chelating the free ionic calcium in the milk is formed, and a pharmaceutically acceptable carrier.
The present invention successfully addresses the shortcomings of the presently known configurations by providing methods of ceasing lactation, causing involution, and treating infection in individual mammary glands of lactating animals without disturbing lactation in remaining glands of the same animal. This allows high producing animals to remain in the herd during treatment. In addition, pharmaceutical compositions provided as part of the present invention reduce the dependence of herdsmen on antibiotics to treat infection, alleviating both the problem of antibiotic resistant mastitic infections, and the perceived problem of widespread agricultural use of antibiotics as a cause of antibiotic resistance in bacteria which infect humans. As a further benefit, the present invention will successfully overcome the problem of persistence of mastitic infection from one lactation cycle to the next. Therefore the present invention is recommended for routine prophylactic use when drying lactating animal.